Pharmaceuticals

Kaposi's sarcoma (KS) is an angioproliferative tumor associated with infection by human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV). Among the viral genes involved in KS pathogenesis, the viral G protein-coupled receptor (vGPCR) plays a key role in oncogenic transformation and angiogenesis, and its sustained activity contributes to tumor development and maintenance. Quercetin (QUE), a naturally occurring flavonoid with well-recognized anticancer properties, has been shown to reduce cell viability and proliferation and to induce apoptosis in vGPCR-transformed endothelial cells. This study further investigated the mechanisms underlying the antineoplastic effects of QUE in vGPCR cells, an in vitro model of Kaposi's sarcoma. Initially, QUE promoted the activation of MAPK signaling pathways in vGPCR cells, increasing the phosphorylation levels of ERK1/2, JNK1/2, and p38, without affecting AKT phosphorylation. In parallel, QUE increased intracellular oxidant levels, followed by lipid peroxidation and DNA damage, supporting a link between oxidative imbalance and genotoxicity. However, increased total glutathione content, and unaltered Gpx4 mRNA expression, suggest the induction of oxidative stress and compensatory antioxidant responses. Furthermore, increased intracellular oxidant levels were associated with MAPK signaling activation, particularly JNK1/2, whereas ERK1/2 and p38 activation may reflect a compensatory response that is insufficient to overcome QUE-induced cytotoxicity. Taken together, these findings indicate that QUE exerts antiproliferative effects in vGPCR cells through a mechanism involving oxidative stress, MAPK activation, and DNA damage, supporting its potential relevance in the study of therapeutic strategies for virus-associated malignancies.

This study evaluated the Delayed Cmax Drug Delivery System (DCMax), an erosion-based controlled- release formulation designed to delay peak plasma concentration (Cmax) and sustain drug release. The tablet matrix contained hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate succinate, and polyethylene oxide. In-vitro dissolution and erosion studies confirmed the delayed release profile. In-vivo performance was assessed in six healthy volunteers by measuring salivary caffeine concentrations after administration of immediate-release caffeine tablets and DCMax tablets. DCMax induced a delayed Cmax of 5 hours compared with 1 hour for the immediate-release formulation, while peak concentrations were lower compared to immediate release. These findings demonstrate that erosion-based drug delivery can achieve delayed release, with potential application in chronotherapy for endocrine disorders.

Background: Endometriosis is an estrogen-driven, inflammatory disorder affecting ~10% of menstruators, causing severe pain. Current treatments that reduce estrogen or inflammation have inconsistent efficacy and poor tolerability. Therapies identified via drug-repositioning methods, which target patient-specific pathways, offer a promising alternative. Simvastatin and primaquine were identified as potential treatments, strongly reversing endometriosis-associated gene expression pathways, and behavioral testing in an animal model showed that these drugs diminish endometriosis-associated pain. This paper examines their effects in human assembloids to provide proof-of-concept validation and insights into their mechanisms of action relevant to endometriosis. Methods: We established human endometrial assembloids using immortalized epithelial (12Z) and stromal fibroblast (iEc) cell lines, as well as primary tissue from patients. Assembloids were generated in 96-well agarose molds with both cell types. We tested ibuprofen, fenoprofen, primaquine, and simvastatin. Following a 24-hour exposure, assembloids and monolayer cultures were harvested and stranded mRNA-seq libraries were sequenced on an Illumina NovaSeqX Plus System. Results: Gene set enrichment analysis showed key pathway reversals: Primaquine reversed the chemical carcinogenesis ROS pathway in assembloids. Simvastatin reversed cytokine-cytokine receptor interaction in both epithelial and stromal cell lines, and the cytoskeleton in muscle cells pathway in stromal cell lines. Fenoprofen reversed the calcium signaling pathway in stromal cell lines. Conclusion: Simvastatin induced the most significant gene expression changes, notably reversing cytokine-cytokine receptor interactions in both cell lines, mirroring findings from our rat model. Despite the small sample size limitation, these experiments highlight the promise of assembloid models to test therapeutic candidates for endometriosis.

Nicotinamide riboside (NR), an endogenous precursor to the essential coenzyme nicotinamide adenine dinucleotide (NAD+), is characterized as safe and effective for longitudinally elevating NAD+ in blood and tissues, when administered orally. Interest in alternative routes of administration of NR has increased in recent years and led to the development of pharmaceutical-grade NR for intravenous and injectable administration. To evaluate the safety and efficacy of repeat daily injections of NR, an open-label, pilot, trial was initiated. Utilizing a 2x2 parallel study design, 39 study participants were randomized to intramuscular (IM) or subcutaneous (SC) injection administration, and 50 or 100 mg of NR, for 3 consecutive days, followed by a 7-day washout (50 mg IM, n=7; 100 mg IM, n=11; 50 mg SC, n=11; and 100 mg SC, n=10). To assess the primary outcome of safety, vitals, blood biomarkers, and adverse event monitoring were employed. Additional assessments included NAD+ pharmacokinetics and subjective participant-reported outcomes. Daily injections of NR were well tolerated and did not result in any unexpected adverse events or experiences. Pain and muscle soreness/tightness more than two minutes after the injection were reported by 44% and 41% of the participants, respectively, regardless of the route of administration or dose. Significant heterogeneity of pharmacokinetic parameters for NAD+ augmentation of whole blood was observed, however an average of the daily percentage increases in NAD+ was consistent across treatment groups, resulting in 9.51, 9.97, 7.83, and 9.05% for 50 mg IM, 100 mg IM, 50 mg SC, and 100 mg SC, respectively. Exploratory participant-reported outcomes suggest potential benefits on fatigue and sleep, with mixed effects on global physical health. Repeat daily injections of NR were well tolerated, and findings warrant investigation in larger, powered, placebo-controlled trials.

Although therapeutic advancements have dramatically improved outcomes for cancer patients in recent decades, advanced cancer remains a significant challenge. There is still an urgent need for new and more effective strategies that minimize toxicity and improve survival outcomes in cancer patients. Natural products from microorganisms are a rich and promising source of bioactive compounds with substantial therapeutic potential in cancer treatment. Recently, B. proteolyticus strain OSUB18, isolated from switchgrass, has been demonstrated to promote plant growth and regulate reactive oxygen species (ROS) production. Here, we investigated the anticancer potential of this plant-associated microbe and demonstrated that its crude extracts exhibited broad-spectrum cytostatic activity across diverse solid tumor cell lines. Importantly, OSUB18 extracts displayed limited cytotoxicity toward normal human mammary epithelial cells and mouse fibroblasts. This potentially selective cytostatic activity was retained within a water soluble, heat-labile, and protease-resistant fraction. The extracts primarily suppressed cell proliferation, accompanied by increased lipid peroxidation, with preliminary evidence suggesting the potential involvement of ferroptosis. These findings suggest that the bioactive component may be a selective cytostatic agent with multifaceted mechanisms of action.

The delivery of small-molecule therapeutics across cellular membranes remains a fundamental challenge in drug development, particularly for compounds whose physicochemical properties limit intracellular bioavailability. Inspired by the low toxicity, biodegradability, and synthetic accessibility of triethanolamine-derived lipids, we investigated a series of C18 aminotriester lipids as excipients for the codelivery of anticancer therapeutics. Four structurally related triester lipids bearing saturated, cis-monounsaturated, trans-monounsaturated, and di-unsaturated C18 tails were synthesized through straightforward esterification reactions with triethanolamine. These lipids were evaluated for their ability to enhance the antiproliferative activity of several anticancer agents, including paclitaxel, doxorubicin, amonafide, homoharringtonine, camptothecin, and SN-38. Across multiple drug classes, the aminotriester lipids exhibited minimal intrinsic cytotoxicity and generally produced limited enhancement of drug potency. However, the fully saturated tris-stearate aminotriester lipid selectively enhanced the antiproliferative activity of amonafide in HCT-116 colorectal cancer cells, particularly at lower concentrations. In contrast, unsaturated lipid analogues displayed negligible enhancement effects, and the activity of the tris-stearate lipid was substantially attenuated in A549 lung adenocarcinoma cells, weakly observed in HT-29 colorectal cancer cells, and absent in CT26 murine colorectal cancer cells. Lactate dehydrogenase release assays indicated that these effects were not associated with membrane lysis or nonspecific cytotoxicity. Further, fluorescence microscopy time course experiments suggest improved membrane trafficking of amonafide when codelivered stoichiometrically with fully saturated C18 aminotriester lipids. Collectively, these findings demonstrate that saturated triethanolamine-based aminotriester lipids may function as selective and biocompatible codelivery excipients for certain small-molecule therapeutics. The observed dependence on both lipid saturation state and cellular context suggests that membrane organization and lipid packing may play important roles in mediating these effects. These results establish a foundation for further mechanistic investigations into aminotriester lipid-assisted small-molecule delivery systems.

Albumin binding can substantially alter the unbound concentration and apparent cellular potency of small-molecule anticancer agents. BAY-1816032 is a selective BUB1 kinase inhibitor with activity in malignant pleural mesothelioma, but its interaction with serum albumin has not been defined. This study combined intrinsic bovine serum albumin (BSA) fluorescence measurements, molecular docking, and cell-viability assays to examine whether albumin association influences BAY-1816032 activity. BAY-1816032 produced pronounced quenching of BSA fluorescence at low drug-to-protein molar ratios, and the quenching response was greater at mildly acidic pH than at mildly basic pH. Docking to BSA (PDB 4F5S) placed the top-ranked pose in Sudlow site I (subdomain IIA), with a Vina score of -7.8 kcal/mol and predicted hydrogen-bond contacts with Tyr149, Arg256, and Asp450. In NCI-H28, NCI-H2052, NCI-H2452, and AB12 mesothelioma cells, supplementation of 10% fetal bovine serum medium with 2% (w/v; 20 mg/mL) BSA shifted BAY-1816032 concentration-response curves to the right. Relative to 10% serum alone, IC50 values increased from 2.4 to 11.1 uM in H28, 3.6 to 22.0 uM in H2052, 2.0 to 4.9 uM in H2452, and 2.4 to 11.1 uM in AB12. These data support an albumin-dependent reduction in the freely available drug fraction and identify site I as the leading computational binding-site hypothesis. Because fluorescence-intensity and docking data do not independently establish a dissociation constant or quenching mechanism, direct unbound-fraction measurements and competitive displacement experiments are required for quantitative confirmation.

Background. Cannabidiol (CBD) products are sold in many forms for many purposes, but it is largely unknown which characteristics of real-world use relate to how users evaluate their experience. Methods. We analyzed an anonymous web-based survey of 377 CBD product users in Japan. We evaluated perceived-effect (0-5) and satisfaction (0-6) scores using main regression models of use frequency adjusted for age, sex, and body mass index. Separate supplementary models examined estimated daily CBD amount and other-cannabinoid co-use, purposes of use, or specific CBD amount-reporting status. Results. Use frequency was positively associated with perceived effects (B = 0.148, 95% CI 0.091 to 0.205) and satisfaction (B = 0.113, 95% CI 0.049 to 0.177; both p < 0.001). Estimated daily amount showed no clear association with either outcome after adjustment, whereas participants who reported specific milligram information had higher scores on both (B = 0.543, p < 0.001 and B = 0.559, p = 0.002). Amount-reporting status was associated with product form (tincture 48.5% vs 30.8%; smoking or inhalation 58.5% vs 75.7%; both FDR-adjusted p = 0.009), whereas no statistically significant group differences were found for age, sex, body mass index, use frequency, or sleep timing. Purposes of use and sleep-timing indicators showed no clear associations with the outcomes. Conclusions. Subjective evaluation of CBD use was more consistently related to how often CBD was used and whether a specific milligram amount was reported than to how much was reportedly taken, and amount-reporting status was related to product form. Specific CBD amount-reporting status may be a useful complementary dimension for characterizing real-world CBD use.

Chronic pain and insomnia are common, each has serious consequences and they often coexist [1-5]. This novel study employed a phenomenological method to explore adults lived experiences of the core and meaning of comorbid insomnia and pain, here conceptualized as COMIP. The empirical data is based on 22 semi-structured interviews with adults with COMIP. Each interview was analysed, developing themes and analytical frameworks for all participants, which were then compared to construct one main analytical framework. The consolidated criteria for reporting qualitative research were used [6]. The essence of COMIP was: A vicious cycle of insomnia and pain driven by four intertwined primary factors with cross effects, which reduced sleep quality and quality of life. Four main themes emerged: Firstly, insomnia with hyperarousal like racing thoughts, and difficulties with finding comfortable sleeping positions due to pain, disturbed sleep and decreased sleep quality. Secondly, daytime symptoms like reduced energy, with reduced daytime function and reduced quality of life. Thirdly, 24-hour hyperarousal, stress and distressing experiences such as suffering. Finally, contextual factors like treatment reduced and/or increased COMIP. The main contribution of this research is three-fold; mapping of the experiences of living with COMIP, the conceptualization of COMIP, and introduction of novel concepts. Our findings indicate that, when evaluating patients with COMIP, there is value in exploring the four COMIP factors. Although, it varies between individuals which factor/s is central to their COMIP experience, we emphasize that COMIP is distressing, and patients are likely to need emotional support and validation. Keywords: COMIP, sleep, phenomenology, disability, rehabilitation, ICF.

Plant-derived flavonoids show measurable anti-dengue activity in cell culture, but their translational value is limited by a narrow therapeutic window: the concentrations that inhibit the virus approach or exceed those that are cytotoxic. Whether nanoparticle formulation can resolve this constraint, rather than simply add potency, remains untested for a chemically defined fraction. Here we show that biogenic silver nanoparticle (AgNP) formation using a flavonoid-enriched fraction of Carica papaya inverts an unusable selectivity profile into a viable one. The unformulated fraction was cytotoxic below the concentrations required for antiviral activity: its 50% cytotoxic concentration (CC50 = 134.9 ug/mL) lay below its 50% effective concentration against dengue virus serotype 2 (DENV-2; EC50 = 254.4 ug/mL), giving a Selectivity Index (SI) of 0.53. Using the same flavonoids as sole reducing and capping agents produced AgNPs (Z-average 128.4 nm; PDI 0.232; zeta potential -28.4 mV) that moved both parameters simultaneously. CC50 rose approximately 8.6-fold to 1,165.74 ug/mL while EC50 fell approximately 8.8-fold to 29.05 ug/mL, raising the SI to 40.12, an approximately 75-fold shift. Time-of-addition analysis localised the effect to the extracellular phase: inhibition was significant under pre-treatment and co-treatment but not after viral adsorption, identifying the AgNPs as entry inhibitors rather than replication inhibitors. Consistent with a serotype-independent physical mechanism, AgNP treatment at 30 ug/mL reduced viral RNA across all four serotypes, using inocula standardised against WHO-traceable NAAT reference reagents. These findings identify capping chemistry, rather than silver content alone, as a determinant of the therapeutic window in phytosynthesised nanoantivirals.

Chemobrain, characterized by cognitive dysfunction, has emerged as a significant health concern following cancer treatment. Doxorubicin, an FDA-approved chemotherapeutic agent, is widely used to treat various cancers. However, its adverse effects raise concerns regarding toxicity in non-cancerous cell types. Microglia, the resident immune cells of the brain, play a central role in maintaining neural homeostasis. Despite this, the mechanisms by which microglia become dysfunctional following doxorubicin exposure remain poorly understood. Understanding the transcriptomic alterations induced by doxorubicin in microglia could provide critical insights into mechanisms underlying its neurotoxic effects. In this study, the transcriptomic response of human microglia to doxorubicin using mRNA sequencing combined with bioinformatic analysis was investigated. Exposure to doxorubicin significantly altered microglial morphology, promoting a shift toward a process-bearing phenotype in human HMC3 cells. This phenotypic change was accompanied by elevated levels of DNA damage and was associated with the induction of cellular senescence. At the gene regulation level, the Illumina-based transcriptomic analysis provided 25,834 gene expression profiling in which 5,669 were differentially expressed. The future analyses revealed that 3,431 (upregulated) and 2,238 (downregulated) were differentially expressed under the treatment of doxorubicin. Genes associated with the p53 signaling pathway and inflammatory responses were upregulated, whereas genes involved in translation, DNA replication, and cell-cycle progression were downregulated. This transcriptional dysregulation was accompanied by differential expression of key transcription factors. This study provides transcriptomic evidence elucidating the genotoxic response to doxorubicin in microglial cells, offering mechanistic insights into chemobrain and highlighting key molecular pathways as potential targets for therapeutic intervention.

Hypertriglyceridemia is commonly managed with lipid-lowering drugs such as statins; however, adverse effects, dose intolerance, and financial barriers may limit their use. Therefore, affordable and potentially safer plant-derived alternatives warrant investigation. This study evaluated the lipid-lowering effects of ethanol extracts of Fucus vesiculosus and Phytolacca berry, administered individually or in combination, in mice fed a butter-enriched diet for 12 weeks. Forty-eight mice were randomly assigned to six groups: normal control (Group 1), butter control (Group 2), statin control (Group 3), Fucus vesiculosus extract (Ext-1; Group 4), Phytolacca berry extract (Ext-2; Group 5), and the combined extracts (Group 6). Body weight, organ-to-body-weight ratios, serum lipid parameters, and histopathological changes were assessed. Group 3 showed the lowest final body weight, while Group 6 demonstrated a comparable body-weight trajectory. Organ-to-body-weight ratios varied among groups, with Group 3 generally showing the highest values and Group 6 comparatively lower ratios for several organs. Group 5 had the lowest total cholesterol concentration, followed by Group 3, whereas Group 6 showed cholesterol levels comparable to the normal control and statin groups. Notably, Group 6 exhibited the lowest serum triglyceride concentration (129.9 mg/dL). Histopathological examination revealed relatively mild hepatic alterations and limited vascular plaque formation in Group 6. The combined extracts demonstrated favorable effects on body-weight gain and serum lipid profiles, particularly triglycerides, with comparatively mild histopathological changes. Further mechanistic, dose-response, toxicity, and long-term studies are warranted before clinical application can be considered.

Introduction: In both humans and experimental animals, obesity is associated with hypothalamic inflammation and neuronal metabolic dysfunction, which contribute to disrupting body energy homeostasis. Although ciliary neurotrophic factor (CNTF) is a neuroprotective cytokine with recognized anti-obesity effects in peripheral metabolic tissues, its role in obesity-associated hypothalamic dysfunction is still poorly characterized. We investigated whether CNTF exerts any direct effects on mHypoE-N41 (N41) hypothalamic neurons and whether it modulates the effects induced by palmitate (PA), an established model of obesity-associated neuronal metabolic dysfunction. Methods: N41 cells were administered CNTF alone or combined with PA to assess whether CNTF affects PA-induced lipotoxic stress. Gene and protein expression was assessed by qRT-PCR, western blotting, and immunofluorescence analysis. Results: In N41 cells, CNTF activated the STAT3, ERK and AMPK signaling pathways. It also upregulated genes involved in mitochondrial metabolism and fatty acid oxidation, including Ucp2, Cpt1 and Pgc1, as well as the proliferation-associated markers Ki-67 and PCNA. PA treatment induced inflammatory and endoplasmic reticulum stress responses and impaired insulin signaling. In PA-treated N41 cells, CNTF significantly attenuated the expression of pro-inflammatory markers Tnf, Mcp1 and Nfkb, restored insulin-induced AKT phosphorylation, and partially reversed PA-induced changes in the expression of metabolic genes Ucp2 and Cpt1. Conclusions: Collectively, these findings indicate that CNTF exerts direct protective metabolic effects on hypothalamic N41 neurons subjected to lipotoxic stress and supports a role for it in the preservation of neuronal metabolic function under obesogenic conditions.

Nicotinamide riboside (NR), a precursor to the essential coenzyme nicotinamide adenine dinucleotide (NAD+), has been extensively studied following oral administration, while the longer-term safety and physiological effects of injectable NR remain less characterized. Following an initial in-clinic phase evaluating repeat intramuscular (IM) and subcutaneous (SC) administration of pharmaceutical-grade NR, participants transitioned to an at-home phase designed to assess the safety, feasibility, and exploratory physiological effects of repeated self-administered SC administration. Participants continued their assigned 50 or 100 mg dose group and self-administered NR subcutaneously 3 times weekly for approximately 90 days. Safety was assessed through adverse event (AE) monitoring, vital signs, and clinical laboratory health measures. Exploratory assessments included markers of mitochondrial bioenergetics, urinary biomarkers of oxidative stress, and participant-reported outcomes related to fatigue, sleep, pain, and quality of life. At-home administration of NR was generally well-tolerated, with no clear dose-dependent safety concerns identified over the study period. Further, participants were able to successfully self-administer repeated SC injections over the study period, demonstrating positive feasibility. Whole-blood NAD+ concentrations remained largely stable over time in both dose groups. MeScreen analysis demonstrated a reduction in GlycoATP at Day 30 and increases in the MitoATP/GlycoATP ratio at Day 30 and Day 90, signifying a preferential shift toward oxidative metabolism. These findings may be indicative of positive metabolic health shifts. Urinary oxidative stress profiling demonstrated heterogeneous temporal responses. Several lipid oxidation markers significantly declined at Day 30 but returned toward baseline at Day 90. Select markers of nucleic acid, nitrative stress, and advanced glycation end-product oxidation demonstrated more sustained reductions. These results for which the majority of measured oxidative stress markers improved suggest that NR injections may exhibit anti-oxidative characteristics. Validated participant-reported sleep and fatigue measures showed improvements. Self-reported facial skin improvement increased descriptively from 11.4% at baseline to 27.8% at Day 30 and 36.8% at Day 90; improvements noticed by others increased from 9.1% to 23.7%, while reported body-skin improvement increased more modestly, from 8.8% to 16.7%. As these outcomes were not designed or powered to establish efficacy or definitive clinical claims, the findings should be considered hypothesis generating and provide preliminary data to inform the selection of outcomes and design considerations for future randomized, placebo-controlled, adequately powered trials of SC NR administration.

Nerve Growth Factor (NGF) supports neuronal survival, plasticity, and growth by binding to p75NTR and TrkA receptors. There is a strong link between NGF and the cholinergic hippocampus, and changes in NGF pathways are observed in Alzheimer's disease (AD). Research shows NGF's neuroprotective and regenerative effects on cholinergic neurons, with positive results in AD models and clinical studies. However, NGF therapy faces challenges: limited blood-brain barrier crossing and dose-dependent pain. To address pain, a mutated NGF called CHF6467 was developed. Thanks to the R100E mutation, it prevents binding to p75NTR while maintaining TrkA affinity, which helps reduce pain side effects. Understanding CHF6467's mechanism remains a challenge. In this context, untargeted metabolomics is effective for uncovering unforeseen responses, making it suitable for assessing CHF6467's overall effects. Building on our previous research and recognizing the importance of amyloid-{beta} peptide as a critical pathological marker of AD, we used NMR-metabolomics to study the effect of CHF6467 on SH-SY5Y cells. The study examined metabolomic profiles of cells treated with CHF6467, with and without A{beta}(1-42), using NMR. Results show CHF6467 influences the metabolic changes caused by A{beta}(1-42), inducing biochemical effects consistent with the promotion of phospholipid precursors, reduced glutamate excitotoxicity, and restored energy metabolism.

BackgroundPremature ovarian insufficiency (POI) is a condition affecting female fertility e.g., in the context of autoimmune disease. A recent proof-of-concept study showed that B-cell depletion with rituximab (RTX) temporarily restored ovarian function in patients with autoimmune POI. However, the underlying immunological mechanisms behind this response is unknown. ObjectivesTo characterize B- and T-cell phenotypes before and after RTX therapy in the above-mentioned patients and associations with autoantibody levels and treatment response. MethodsCryopreserved cell samples were available from six autoimmune patients with POI, four of whom were positive for autoantibodies against steroidogenic enzymes (21OH, 17OH and/or SCC). Patients received RTX and blood samples were collected at baseline, second infusion (2 weeks), 3, 8 and 12 months. Peripheral B- and T-cell phenotypes were analysed using spectral flow cytometry and correlated with autoantibody levels. ResultsAt baseline, patients displayed signs of B-cell activation by alteration in subset composition compared to matched controls. Upon closer analysis, patients positive for steroidogenic enzyme autoantibodies had significantly increased frequency of CXCR3+switched memory (SWM) B-cells than autoantibody-negative patients, which correlated positively with anti-17OH and-SCC autoantibody levels. In parallel, increased Th1 cell frequencies were also observed in the same patient group. Longitudinal analyses showed expected B-cell depletion followed by repopulation after RTX. Changes in T-cell subsets were observed at 8 months with reduced frequencies of Th1 and Tfh cells. ConclusionAutoimmune POI displayed altered B- and T-cell subsets implicating cell crosstalk, immune cell infiltration to sites of inflammation such as ovaries, and presence of Th1-driven immunopathogenesis. Key messagesO_LIIncreased CXCR3+SWM B cells and Th1 cells were found in patients with autoimmune POI who were positive for autoantibodies against steroidogenic enzymes. C_LIO_LICXCR3 expression on SWM B cells positively associated with anti-17OH and anti-SCC autoantibody levels. C_LIO_LIRTX induced rapid B-cell depletion followed by repopulation and a delayed T-cell effect at 8 months with reduced Th1 and Tfh cell frequencies. C_LI Capsule summaryRTX has recently been shown to restore fertility in autoimmune POI. Lymphocyte profiling identified Th1 bias accompanied by CXCR3+memory B cells that correlate with autoantibody levels. B-cell depletion appears to temporarily alleviate ovarian autoimmunity and ameliorate physiological function.

In 1998 we predicted percent human intestinal absorption (HIA) for 86 drug and drug-like compounds from six molecular descriptors chosen by a genetic algorithm with a small neural network serving as the fitness function. That result rested on one division of the compounds into three sets: training, cross-validation and external prediction. Repeating the division shows it was a rather fortunate one. An extraction is one random draw from a wide distribution, so we repeat every experiment 250 times and report the result as a distribution. Across 250 stratified extractions, 14 match the published cross-validation error and only one matches the published external prediction error. Reconstructed this way, the published six descriptors give a mean extraction error roughly twice the external prediction value we reported in 1998 (Table 1). We then applied the same six descriptors to a larger set of 780 compounds from a public compilation. Under the 1998 network they give 22.50 %HIA units on training compounds against 25.77 on extracted compounds, whereas on the 86 they give 13.43 against 30.39. More compounds close the gap between fitting and prediction. Four datasets are modeled here: 780 small molecules with HIA measured in man, 3,771 small molecules and 1,281 cyclic peptides measured across Caco-2 cell monolayers, and 7,298 cyclic peptides measured across artificial membranes. Each is its own prediction problem, and one procedure is applied to all four. A genetic algorithm chooses which descriptors to use and how many, and a random forest grades each choice on compounds its own trees never saw. The descriptors chosen most often across the 250 extractions form one consensus set for each dataset. For absorption, 25 consensus descriptors under a random forest reach an extraction rms of 20.47 %HIA units at R 0.76. On the same compounds and the same extractions, the six 1998 descriptors under the 1998 network reach 25.77 at R 0.59. The four models are served at predicthia.ai, where a structure can be drawn or pasted, scored against its training set for similarity, and the training data with fitted models downloaded. Each model served is its fixed consensus set fitted on every compound in its dataset; the accuracy quoted beside a prediction comes from evaluating that same set across 250 extractions (Table 3). The fixed sets hold 25 descriptors for absorption, 55 for Caco-2 small-molecule permeability, 50 for Caco-2 peptide permeability and 25 for artificial-membrane permeability. Keywords: human intestinal absorption; QSPR; genetic algorithm; descriptor selection; cyclic peptides; membrane permeability; oral bioavailability; repeated extraction

Cardiovascular diseases (CVD) are the leading cause of morbidity and mortality worldwide. This clinical burden is predominantly attributable to atherothrombotic events resulting from platelet hyperactivation, a process integral to the pathophysiology of atherosclerosis. Cilostazol (CLZ) is a clinically established phosphodiesterase-IIIA inhibitor with antiplatelet and vasodilatory activity. However, its extremely low aqueous solubility results in dissolution-limited and variable oral absorption, while dose-dependent adverse effects and cardiovascular safety restrictions may constrain its clinical use. Lipid-polymer hybrid nanoparticles (LPHNPs) represent a highly promising drug delivery system to address these biopharmaceutical limitations. However, their interactions with blood components remain poorly understood, with surface charge among the most controversial physicochemical parameters implicated in platelet toxicity. Here, we applied an integrated ex vivo and in silico screening strategy to design LPHNPs based on poly(lactic acid) and poly(ethylene glycol)-modified lipids displaying positive (amine), nominally neutral (methoxy), and negative (carboxyl) surface charges to systematically identify safe boundaries at the nano-human platelet interface and evaluate their suitability as CLZ carriers. Positively charged nanoparticles precipitated immediately during self-assembly due to uncontrolled ionic pairing and were therefore structurally unviable. Neutral nanoparticles maintained a stable size (~113 nm hydrodynamic diameter) but unexpectedly induced spontaneous platelet aggregation, limiting their applicability. In contrast, negatively charged nanoparticles exhibited exceptional colloidal stability (hydrodynamic size of ~120 nm and zeta potential of -48 mV), achieved significant inhibition of ADP-induced platelet aggregation, and adhered to the platelet surface without inducing activation or membrane disruption. Steered molecular dynamics simulations, performed under identical non-equilibrium pulling conditions, showed the highest interfacial resistance to CLZ extraction for the negatively charged model, qualitatively consistent with the sustained CLZ release profile observed in vitro. These findings highlight the importance of precise surface charge tuning in the development of safe cardiovascular nanomedicines based on LPHNPs, emphasizing the need for comprehensive platelet compatibility testing before further biological evaluation.

Introduction: Preterm birth is often associated with fetal membrane (FM) inflammation. Opioid-use disorder during pregnancy is a major public health issue as all opioids carry a risk for preterm birth. While maintenance therapy with methadone improves maternal outcomes, it also elevates the risk of preterm birth. We previously reported that the opioid, buprenorphine, induced human FM sterile inflammation. The goal of this study was to investigate if methadone also induced FM inflammation and to determine the mechanisms involved. Methods: Human FM explants were treated with or without methadone in the presence or absence of inhibitors to TLR4/MD2, TLR7, TLR8, NLRP3, the -opioid receptor, p65 NF{kappa}B, p38 MAPK, ERK or JNK. Supernatants were measured for cytokines/chemokines by ELISA. Tissues were analyzed by Western blot for NF{kappa}B and MAPK activity, and by RT-qPCR for the TLR7/8-activating miRs: miR-21a, miR-29a, miR-146a-3p, Let-7b. Pregnant C57BL/6 mice were injected with PBS or methadone on E15.5 and 6hrs later FMs were analyzed by RT-qPCR. Results: Methadone elevated human FM explant secretion of IL-1{beta}, TNF, MIP-1, GM-CSF, and PGE2, and expression of miR-21a. In vivo, methadone elevated mouse FM expression of MIP1A, KC, and COX2. TLR4/MD2 inhibition had no effect on human FM inflammation in response to methadone. NLRP3 inhibition reduced methadone-induced FM IL-1{beta}, TLR7 inhibition reduced methadone-induced FM TNF, and inhibition of either TLR8 or the -opioid receptor reduced methadone-induced secretion of TNF, MIP-1, GM-CSF, and PGE2, but not IL-1{beta} Methadone increased human FM activity of p65 NF{kappa}B, p38 MAPK, JNK, and ERK and inhibition of these signaling pathways collectively reduced methadone-triggered FM inflammation. Finally, naloxone reduced most of the methadone-induced FM inflammatory response. Conclusion: Methadone induces sterile inflammation in human and mouse FMs. In human FMs this inflammation is mediated through activation of innate immune TLR7, TLR8, and NLRP3; the -opioid receptor; and NF{kappa}B/MAPK signaling. These findings provide further insight into the mechanisms underlying the association between opioid use during pregnancy and preterm birth.

Ovarian cancer stands out as one of the most lethal gynecological cancers among women in the United States. While patients with ovarian cancer often show positive responses to treatment, it is common for the cancer to reappear and develop resistance to treatment, so it is important to explore and create novel combinations to increase patient survival rates. ATRi (Ataxia telangiectasia and Rad3-related inhibitors) and ONC201 monotherapies have shown efficacy in ovarian cancer in preclinical models. We hypothesized that combining ATRi with ONC201 would enhance this efficacy. ONC201 induces apoptosis through the TRAIL-pathway following activation of the integrated stress response and inactivation of Akt which is upregulated in ovarian cancer. Six human ovarian cancer cell lines were treated alone and with the novel drug combination of ceralasertib (ATRi) and ONC201. We found an IC50 range of 0.88-20.16 microM for ONC201 and IC50 range of 0.55-54.75 microM for ceralasertib. The combination of ONC201 plus ceralasertib resulted in PARP cleavage consistent with apoptosis along with reduction in Bcl-XL, Xiap and p-Akt expression after 72 hours of treatment. Cytokine profiling showed a decrease in markers of inflammation and tumor progression. Our data provide evidence of synergy from the combination of ATRi ceralasertib and ONC201 against Ovarian cancer cells that merits further investigation including clinical translation.